The present invention relates generally to analogs of GM-CSF and IL-3 proteins, and particularly to the construction of fusion proteins comprising CM-CSF and IL-3.
The differentiation and proliferation of hematopoietic cells is regulated by secreted glycoproteins collectively known as colony-stimulating factors (CSFs). In humans, these proteins include granulocyte-macrophase CSF (GM-CSF), which promotes granulocyte and macrophage production from normal bone marrow, and which also appears to regulate the activity of mature, differentiated granulocytes and macrophages. IL-3 (also known as multi-CSF) also stimulates formation of a broad range of hematopoietic cells, including granulocytes, macrophages, eosinophils, mast cells, megakaryocytes and erythroid cells. GM-CSF and IL-3 thus have considerable overlap in their broad range of biological activities. Other CSFs have a more restricted range of activity, macrophage CSF (M-CSF) stimulating almost exclusively macrophage colony formation, and granulocyte CSF (G-CSF) stimulating primarily granulocyte colonies. Although GM-CSF and IL-3 have distinct amino acid sequences, preclinical studies indicate that they may be useful to treat various cytopenias, and to potentiate immune responsiveness to infectious pathogens, and to assist in reconstituting normal blood cell populations following viral infection or radiation or chemotherapy-induced hematopoietic cell suppression. The genes encoding GM-CSF and IL-3 are located on the same chromosome in mouse and man and the expression of the genes is linked in some cells, such as activated T lymphocytes (Kelso et al., J. Immunol. 136:1718, 1986; Yang et al., Blood 71:958, 1988; Barlow et al., EMBO J. 6:617, 1987).
Short-term experiments have demonstrated that the simultaneous combination of GM-CSF and IL-3 was more effective than either GM-CSF or IL-3 alone in increasing cycling rates and numbers of marrow hematopoietic progenitor cells in vitro in lactoferrin-treated mice (Broxmeyer et al., Proc. Natl. Acad. Sci. USA 84:3871, 1987). No such synergy has been observed in vivo for simultaneous administration of GM-CSF and IL-3, although clinical studies have shown that the consecutive administration of recombinant human IL-3 and recombinant human GM-CSF was more effective in raising white blood cell counts in normal cynomolgus monkeys than either GM-CSF or IL-3 along (Krumwieh et al., Behring Inst. Mitt. 83:250, 1988; Donahue et al., Science 241:1820, 1988).
The biological activities of GM-CSF and IL-3 are mediated by binding to specific cell surface receptors expressed on primary cells and in vitro cell lines. GM-CSF and IL-3 each bind to their respective receptor, resulting in transduction of a biological signal to various immune effector cells. Recent studies of the characteristics and distribution of the receptor for IL-3 on the human myelogenous leukemia cell line KG-1 and human pre-B cell line JM-1 indicate that a subclass of receptor exists which also binds GM-CSF (Park et al., J. Biol. Chem. 264:5420, 1989). These studies showed that human GM-CSF is capable of almost completely inhibiting the binding of .sup.125 I-IL-3 to KG-1 cells and, conversely, that IL-3 is capable of substantially inhibiting binding of .sup.125 I-GM-CSF to the same cells. This direct competition between GM-CSF and IL-3 for a single cell surface receptor indicates that a single receptor is capable of binding both GM-CSF and IL-3. Although is not yet clear whether the heterogeneity in IL-3 and GM-CSF binding is due to the existence of a receptor molecule which is distinct from that which binds IL-3 alone or GM-CSF alone, or whether IL-3 and GM-CSF receptors may exist as multisubunit complexes, composed of different ratios of IL-3 and GM-CSF binding proteins, the receptor(s) will be referred to herein as the GM-CSF/IL-3 receptor.